Interpretive Summary: New innovations in DNA sequencing technologies that produce high volumes of quality sequence data are redefining approaches to genetic research. We report one of the first uses of 454 Sequencing in rainbow trout to discover unique genes expressed in a single rainbow trout. In this single experiment, roughly seven times more sequence data was generated by a single laboratory in months compared to several years of sequencing by international collaborators using previous methods. This data set has been made publicly available for use by the rainbow trout genome research community and has dramatically increased the volume of data available for this species.

Technical Abstract:
Background: Rainbow trout is an important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and the evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number of EST (258,973) sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation.
Results: High-throughput deep sequencing of a rainbow trout double-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344bp, a total of 447 million bases. De novo assembly of the sequences yielded 151,847 Tentative Consensus sequences (TCs) (Average length 662 nt) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (Average length 758 nt) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed similarity to transcriptomes of other fish species with known genome sequences, suggesting a genome-wide representation of the rainbow trout transcriptome sequence.
Conclusion: The 454 library significantly increased the suite of the ESTs available for rainbow trout allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, for digital gene expression and proteomic research.